1. Field of the Invention
This invention is directed to compositions comprising an effective amount of cyclosporine in combination with a fatty acid component comprising a fatty acid of the omega-3 family, and to a method for mediating the nephrotoxic effects of cyclosporine comprising administering said cyclosporine in combination with said fatty acid component.
2. Description of the Background Art
Cyclosporine is a cyclic, nonwater-soluble, highly nonpolar molecule composed of 11 amino acids. The compound is a promising immunosuppressive agent which is derived from soil fungus (Calne et al., Transplant Proc. 13:349-358 (1981); Ferguson et al., Surgery 92:175-182 (1982); Starzl et al., Gynecol. Obstet. 151:17-26 (1980)). The drug is now widely used for prolonging the function of various transplanted organs. Its immunosuppressive effects selectively inhibit T-cell function, allowing survival of allografts without myelosuppression, i.e., heart transplants, Myers et al., N. Eng. J. Med. 311:699 (1984).
In addition to its use in allograft recipients, recent clinical trials have been or are being undertaken to examine the efficacy of cyclosporine in the treatment of a wide variety of autoimmune diseases, including polymyositis, systemic lupus erythematosis, rheumatoid arthritis, and early insulin dependent diabetes see relevant chapters in: Cyclosporine in Autoimmune Diseases, ed. Schindler, R., Springer-Verlag, Berlin (1985), particularly von Graffenreid, B., et al., pp. 59-73).
Cyclosporine is a lipophilic molecule with a molecular weight of 1202 daltons. When the drug is dissolved in olive oil or a special solution prepared by the manufacturer, bioavailability and absorption are maximized. The drug readily binds to plasma proteins and has a terminal half-time of 24 hours. It is highly metabolized in the liver, with biliary excretion being the major route of elimination (Beveridge, T., Cyclosporine A:, Proceedings of the International Symposium, Cambridge, White D. J., ed., pages 35-44 (1982)). In addition to its immunosuppressive characteristics, the drug also has interesting anti-schistosome and anti-malarial activities (Kolata, Science (Washington, D.C.) 221:40-42 (1983); Sanches et al., First Int'l. Montreux Conf. on Biol. Rhythms and Medications, Montreux, Switzerland, Mar. 26-30, 1984. Pergamon Press, Oxford (in press).
In spite of its great promise as an immunosuppressive, however, its use is somewhat limited, both by its association with infection and also because of hepatic and renal toxicities (Ryffel, OL 27-400: "Summary of Toxicity Data," Sandoz, Basel, Switzerland (1981)).
Clinical use of cyclosporine is associated with reversible, dose-related increases in blood urea nitrogen (BUN) and serum creatinine levels and depression of creatinine clearance. Some nephrotoxicity is reported to occur in almost 80% of renal transplant patients using cyclosporine (Kahan, B. D., Dial. Transplant. 12:620-30 (1983)). Often the urea nitrogen level is disproportionately increased relative to the serum creatinine level.
Frequent side effects of cyclosporine treatments in various autoimmune diseases include nephrotoxicity, hypertension, hyperkalemia, hyperuricemia, hepatoxicity, anemia, hypertrichosis, gingival hyperplasia, gastrointestinal intolerance, tremor, and paresthesia. von Graffenried, B., et al., in Cyclosporine in Autoimmune Diseases. R. Schindler, ed., Springer-Verlag, Berlin, pp. 59-73 (1985). Of these, the most commonly reported adverse effect is nephrotoxicity.
Bennett, W. M., et al., Ann. Int. Med. 99:851-854 (1983), have pointed out the substantial nephrotoxic potential accompanying cyclosporine therapy in patients receiving kidney, heart, bone marrow, and liver transplants. Acute cyclosporine nephrotoxicity is dose-dependent, correlated with cyclosporine levels in blood or plasma, Kahan, B. D., et al., Transplantation 34:36 (1982), and is reversible after dose reduction, Verani, R. R., et al., Am. J. Kidney Dis. 4:185 (1984), or after cessation of cyclosporine therapy, Chapman, J. F., et al., Lancet I:28 (1985).
Acute cyclosporine nephrotoxicity is morphologically correlated with tubular lesions characterized by inclusion bodies, isometric vacuolization and microcalcification, Mihatsch, M. J., et al., Transplant Proc. 15:2821 (1983). Various hypotheses have been proffered to explain the decline in glomerular filtration rate, as evident by the rapid increase of serum creatinine in cyclosporine-treated patients. These include stimulation of tubuloglomerular feedback, Gutshe, H. U., et al., Ninth Int. Congress of Nephrology, Los Angeles, June 1984, Abstract No. 475A (1984), and disturbance of the microcirculation through interaction of cyclosporine on local prostacyclin synthesis, Neild, G. H., et al., In B. D. Kahan, ed., Cyclosporine, Gruen & Stratton, Orlando, Fla. page 182 (1984).
By contrast to the mode of cyclosporine therapy used in transplant patients, patients with autoimmune diseases often receive lower initial dosages of cyclosporine for longer periods of time. von Graffenried et al., presented data extracted from case report forms of ongoing clinical studies of patients suffering from multiple sclerosis, rheumatoid arthritis, diabetes mellitus type I, uveitis posterior, primary biliary cirrhosis, endocrine ophthalmopathy and systemic lupus erythematosus. These data related to renal function in patients on continuous cyclosporine therapy for up to 24 months and to reversibility of nephrotoxic effects in patients after discontinuing cyclosporine treatment. They reported that cyclosporine induces an increase in serum creatinine within the first two weeks of therapy, and that the steepest decline in renal function occurs within the first three months of chronic treatment, with the mean reduction in creatinine clearance (CRCL) being 14% at month six. Only slight further CRCL impairment occurred after month six, and no relevant further deterioration was reported up to month 24 of cyclosporine therapy, although data for this duration of treatment were limited. The extent of nephrotoxicity was related to cyclosporine dosage and to cyclosporine levels, and may have been age-related. The authors concluded that these factors probably interacted in patients with rheumatoid arthritis, who also show above-average nephrotoxicity. Patients having below-average baseline renal function, however, showed only little and stable renal dysfunction despite average clinical dosage of cyclosporine. The authors observed that cyclosporine-induced renal dysfunction markedly improves after reduction of cyclosporine dose with subsequent lower creatinine levels, and is completely reversible within two months after stopping cyclosporine therapy. Similar reversibility of cyclosporine-induced nephrotoxicity has been reported in diabetes mellitus type I patients (Stiller, C. R., et al., Science 223:1362 (1984)) and in ocular inflammatory disorders of autoimmune origin (palestine, A. G., et al., Am. J. Med. 77:652 (1984)).
In contrast to the reversibility of renal dysfunction induced by chronic cyclosporine therapy in the treatment of autoimmune diseases, progressive and possibly irreversible cyclosporine-induced deterioration of renal function has been described in heart transplant patients (Myers, B. D., et al., N. Eng. J. Med. 311:699 (1984)). Possible irreversible histological findings in kidneys of transplant patients given cyclosporine therapy have also been published (Mihatsch, M. J., et al., Transplant Proc. 15:2821 (1983); Myers, B. D., et al., N. Eng. J. Med. 311:699 (1984)). And, in fact, von Graffenreid et al., supra, noted that, although the data from cyclosporine-treated patients suffering from autoimmune diseases seem to demonstrate full reversibility of cyclosporine-induced acute nephrotoxicity, a very slowly progressive chronic nephropathy could not be excluded, since the parameter used to assess renal function (serum creatinine) is not sensitive enough to detect early nephron loss, and because of the small amount of data for patient treatment beyond one year.
It will be apparent from the preceding discussion that deteriorization of renal function is a major side effect which reduces the practical clinical therapeutic efficacy of cyclosporine treatment for transplant and non-transplant patients. The correlation of cyclosporine dose (and levels) with nephrotoxicity suggests that cyclosporine levels need to be maintained within a very narrow therapeutic range, i.e., low enough to minimize nephrotoxicity but high enough to accomplish immunosuppressive therapeutic objectives. For example, when cyclosporine is administered with the object of avoiding allograft rejection, a steady-state trough level of less than 200 ng/ml probably is not immunosuppressive enough to avoid rejection, whereas nephrotoxicity and other side effects occur more frequently at concentrations greater than 400 ng/ml. Such a narrow therapeutic window is difficult to maintain in clinical practice. Bennett, W. M., et al., Ann. Int. Med.
99:851-854 (1983). Furthermore, it has been suggested that any benefit derived from reduced rejection of renal allografts may be more than offset over the long term by chronic nephropathy induced by the cyclosporine therapy itself. Myers, B. D., et al., N. Eng. J. Med. 311:699-705 (1984). This same concern has been expressed where cyclosporine has been used to suppress the immune inflammation associated with autoimmune diseases, id., because of the risk of inducing severe chronic nephropathy. As a result of these concerns, Myers et al., supra, have also expressed the need for measures that would widen the margin of safety between the dose of cyclosporine required to achieve effective immunosuppression and the dose likely to cause renal damage.
While the mechanism of renal dysfunction is unclear, increased renal synthesis of thromboxane has been demonstrated during the progression of immune-mediated and non-immunologic induced models of renal injury. Lianos, E. A., et al., J. Clin. Invest. 72:1439-1448 (1983); Okegawa, T., et al., J. Clin. Invest. 71:8-90 (1983); Purkeroon, M. L., et al., Kid. Inter. Abstr. 25:251 (1984); Remuzzi, G., et al., Kid. Inter. Abstr. 25:217 (1984); Ichikawa, I., et al., Kid. Inter. Abstr. 25:231 (1984). Thromboxane, a prostanoid, is a metabolite of arachidonic acid derived from the cyclooxygenase pathway. The other prostanoids are the prostaglandins and prostacyclins. The prostanoids are potent mediators generated during immunologically related inflammatory events, and are capable of profoundly changing renal hemodynamics. Morley, J., in Lymphokines, E. Pick, ed. Academic Press, New York, 4:377-391 (1981); Lewis, G. P., Br. Med. Bull. 39:243-248 (1983): Dunn, M. J., in Renal Prostaglandins M. J. Dunn, ed., Williams & Wilkins, Baltimore, pp. 1-74 (1983). Prostanoids and eicosanoids, which are arachidonic acid metabolites, are synthesized by cells according to immediate need and are not stored in significant amounts for later release. Harrison's Principles of Internal Medicine, 10th ed., pp. 482-487 (1983).
Kawaguchi, A., et al., Transplantation 40(2):214-216 (1985), found that excretion of thromboxane B2, a urinary degradation product of thromboxane A2, is strongly correlated with serum cyclosporine levels in rats. The authors conclude that high cyclosporine doses are associated with increased synthesis of thromboxane B2 from renal or extrarenal sources. It is noted that, although clinical toxicity of cyclosporine bears a resemblance to the pathogenic properties of thromboxane A2, it is unclear whether the observed increase in thromboxane B2 synthesis is linked to cyclosporine-induced nephrotoxicity. Cyclosporine has also been reported to induce increased formation of prostaglandins of the E series (PGE) in cultured human monocytes. Whisler, R. L., et al., Transplantation 38:377-381 (1984). The authors note that this increased PGE formation requires cyclooxygenase activity, and suggest that this is mainly mediated through greater availability of endogenous arachidonic acid to the cyclooxygenase pathway.
Human kidney allograft rejection has been shown to be associated with an early increase in urinary excretion of immuno-reactive thromboxane B2 (iTXB.sub.2) (Foegh, M. L., et al., Transplantation Proc. 16(6):1606-1608 (1984)) and has been suggested as an immunologic monitor in kidney transplant patients (Foegh, M. L., et al., Transplantation proc. 16(6):1603-1605 (1984)). Khiabadi, B. S., et al., Transplantation 39(1):6-8 (1985), report that increases in urinary iTXB2 are associated with heterotropic cardiac allograft rejection in a rat model. The authors note that the precise relationship of urinary iTXB2 through the rejection process is still conjectural and remains to be ascertained.
Active metabolites of arachidonic acid are formed by one of two synthetic pathways--the cyclooxygenase or the lipoxygenase system. The products of the cyclooxygenase pathway--the prostaglandin, prostacyclins, and thromboxanes--are collectively termed prostanoids. The term "eicosanoids" includes the products of the lipoxygenase pathway--5-hydroxyeicosatetraenoic acid and leucotrienes--and the prostanoids.
The initial synthetic step of both pathways involves cleavage of arachidonic acid from the phospholipid plasma membrane of cells. Free arachidonic acid can then be metabolized by the cyclooxygenase or lipoxygenase pathway. The first product of the cyclooxygenase pathway is the cyclic endoperoxide PGG.sub.2, which is then converted to PGH.sub.2. These are the key intermediates in the formation of the classical prostaglandins (PGA.sub.2, PGD.sub.2, PGE.sub.2, and PGF.sub.2 -alpha), prostacyclin (PGI.sub.2) and thromboxane A.sub.2 (TXA.sub.2). The first product of the lipoxygenase pathway is hydroperoxeicosatetraenoic acid (HPETE) which is an intermediate in the formation of 5-hydroxeicosatetraenoic acid (HETE) and the leukotrienes (LTA, LTB, LTC, and LTD). It is known that two fatty acids other than arachidonic acid--3,11,14-eicostriaenoic acid (dihomo-gamma-linolenic acid) and 5, 8, 11, 14, 17-eicosapentaenoic acid--can be converted to metabolites closely related to the prostanoids and eicosanoids. The products of these different fatty acid substrates are distinguished by their subscripts: the subscript 1 is given to products of dihomo-gamma-linolenic acid; the subscript 2 is given to arachidonic acid products; and products of 5,8,11,14,17-eicosapentaenoic acid are given the subscript 3. The subscripts additionally designate the number of double bonds between carbon atoms in the side chain of the products.
Arachidonic acid metabolites are rapidly catabolized in vivo. The E and F series prostaglandins are chemically stable, yet are almost completely degraded in a single pass through the liver and lungs. Thus, virtually all non-metabolized PGE measurable in urine derives from renal and seminal vesicle secretion, whereas PGE metabolites in urine represent PGE synthesis by other organs. PGI.sub.2 and TXA.sub.2 are chemically unstable and are also rapidly catabolized. PGI.sub.2 is converted to 6-keto-PGF.sub.1 -alpha, and TXA.sub.2 is converted to TXB.sub.2. Both PGI.sub.2 and TXA.sub.2 are short-lived in vivo, and measurement of their inactive metabolites is the common method used to provide an index of their formation rates.
Arachidonic acid metabolites are postulated to play a role in the pathology of a number of diseases, including hypercalcemia of malignancy, bone resorption in rheumatoid arthritis and dental cysts, Bartter's syndrome, diabetes mellitus, essential hypertension, patent ductus arteriosus, peptic ulcer disease, dysmenorrhea, and asthma.
Several arguments support a relation between arachidonic acid metabolites and the inflammation response: endogenous prostaglandins are released in parallel by histamine and bradykinin; several arachidonic acid metabolites are known to cause vasodilation and hyperalgesia; prostaglandins are present in areas of inflammation, polymorphonuclear cells release PGE during phagocytosis and PGE is a chemotactic for leukocytes; increased vascular permeability, which results in local edema, is caused by some arachidonic acid metabolites; PGE-induced vasodilation is not abolished by atropine, propranolol, methylsergide, or antihistamines, which are known to antagonize other putative inflammatory response mediators, suggesting a direct inflammatory effect of PGE; arachidonic acid metabolites cause pain in animal models and hyperalgesia or increased sensitivity to pain in humans; PGE causes fever after injection into the cerebral ventricles or into the hypothalamus of experimental animals; and pyrogens cause increased concentrations of prostaglandins in cerebrospinal fluid, but prostaglandin synthesis inhibitors decrease fever and decrease release of prostaglandins into cerebrospinal fluid.
Metabolites of arachidonic acid also have a postulated role in the immune response. It is known that small amounts of PGE suppress stimulation of human lymphocytes by mitogens such as phytohemagglutinin, leading to the suggestion that these substances act as negative modulators of lymphocyte function, perhaps by a negative feedback control mechanism. Sensitivity of lymphocytes to the inhibiting effects of PGE.sub.2 increases with age, and indomethacin augments lymphocyte responsiveness to mitogens to a greater degree in the elderly. Lymphocytes cultured from patients with Hodgkin's disease release more PGE.sub.2 after the addition of phytohemagglutinin, and lymphocyte responsiveness is enhanced by indomethacin. When suppressor T cells are removed from the cultures, the amount of PGE.sub.2 synthesized is diminished, and the responsiveness of the lymphocytes from the Hodgkin's patients and controls is no longer different. Depressed cellular immunity in patients with Hodgkin's disease may be the result of PGE inhibition of lymphocyte function. A general discussion of arachidonic acid metabolites relevant to medicine is presented in Harrison's Principles of Internal Medicine, 10th ed., pp. 482-487 (1983).
The obligatory precursor of arachidonic acid is linoleic acid (C18:2 omega-6). Linoleic acid is a polyunsaturated fatty acid of the omega-6 family. The omega number indicates the location of the first double bond counting from the methyl end of the fatty acid. The other two major unsaturated fatty acid families are the oleic acid (omega-9) family and the linoleic acid (omega-3) family. The three fatty acid families are not metabolically interconvertible. The major metabolite of oleic acid is eicostrienoic acid (C20:3 omega-9). The major omega-3 (linoeic) acid family metabolites are eicosapentaenoic acid (C20:5 omega-3) and docosa-hexaenoic acid (C22:6 omega-3). The principal foot sources of linoleic and linolenic acids are seeds and leaves. The major omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, however, are synthesized by phytoplankton, which form the bottom of the marine food chain. As a result, fish, and especially fish oil, are enriched with omega-3-fatty acids, especially eicospentaenoic acid and docosahexaenoic acid. The omega-6 and omega-3 fatty acid families cannot be synthesized de novo by humans, and are regarded as essential fatty acids.
Other polyunsaturated fatty acids besides arachidonic acid may serve as substrates for prostaglandin synthesis. For example, dihomogamma-linolenic acid (DHLA) (C20:3 omega-6) acts as a substrate for prostaglands of the "1" series, such as the classical prostaglandin PGE.sub.1. Willis, A. L., Nutr. Rev. 39:289-301 (1981). Eicosapentaenoic acid (C20:5 omega-3) is the substrate for prostaglandins of the "3" series and, under certain conditions, leads to the production of thromboxane A.sub.3, PGA.sub.3, and PGI.sub.3. While feeding of linolenic acid (C18:3 omega-.sub.3) does not lead to significant increases of eicosapentanoic acid in adult human plasma (Dyerberg, J., et al., Lancet 1:199 (1980)), feeding of marine foods rich in eicosapentaenoic acid leads to rapid incorporation of this fatty acid into both platelet and endothelial cell membranes. See, e.g. Sless, W., et al., Lancet 1:441-441 (1980); Sanders, T. A. B., et al., Lancet 1:1189 (1980); and references cited in Goodnight, S. H., et al., Arteriosclerosis 2:87-113 (1982). In reviewing the effects of dietary polyunsaturated fatty acids of the various fatty acid families, Goodnight et al. concluded that feeding omega-3 fatty acid-rich fish oils to humans leads to a reproducible prolongation of the bleeding time, inhibition of platelet aggregation by ADP and collagen, as well as a decrease in platelet retention on glass beads. In some settings, the authors note, there may also be a reduction in platelet count. The authors conclude that ingestion of dietary fish oils containing the omega-3 fatty acid eicosapentaenoic acid may have profound effects on platelet or vessel composition and function. Cellular phospholipid concentrations of arachidonic acid are decreased, bleeding time prolonged, and various in vitro tests of platelet function are inhibited. One explanation offered by the authors for the platelet inhibition is the significant reduction in platelet thromboxane synthesis.
Ingestion of high levels of dietary fish oils may lead to undesirable side effects. For example, some fish oils contain high levels of cetoleic acid (C22:1 omega-11), an isomer of erucic acid (C22:1 omega-9). High levels of erucic acid are known to cause transient myocardial lipodosis and fibrosis in experimental animals. The Food and Agriculture Organization of the United Nations, Joint FAO/WHO Report, FAO Food & Nutrition Paper, No. 3, Rome, Italy (1977). Feeding high levels of fish oil also leads to the development of yellow fat disease in experimental animals. This disease is associated with vitamin E deficiency, which may be exacerbated by the highly unsaturated nature of omega-3 fatty acids. Garton, G. A., et al., Biochem. J. 50:517-524 (1952). Fish oil feeding affects platelet function, increasing bleeding times, and leads to thrombocytopenia in humans. All this suggests that is may be impractical or even unsafe for humans to ingest very high amounts of dietary fish oils, or to rely solely on fish oil as a lipid source. On the other hand, studies of human populations which historically consume high levels of omega-3 fatty acids, particularly the coastal Eskimos of Greenland, suggest that, aside from prolonged bleeding times and thrombocytopenia, there are no significant adverse effects of a high fish oil diet. Bang, H. O., et al., Acta. Med. Scan. 192:85-94 (1972); Bang, H. O., et al., Acta. Med. Scan. 200:69-73 (1976; Dyerberg, J., et al., Lancet 2:117-119 (1978).
Kelley, V. E., et al., J. Immunol. 134(3)1914-1919 (1985), supplemented the diet of MRL-1pr mice with fish oil as the exclusive lipid source and reported that this suppressed autoimmune lupus. The marine oil diet decreased lymphoid hyperplasia regulated by the 1pr gene, prevented increases in macrophage surface Ia expression, reduced formation of circulating retroviral gp70 immune complexes, delayed the onset of renal disease, and prolonged survival in these mice, as compared to mice given safflower oil as a lipid source. The authors postulate that the unique fatty acids, eicosapentaenoic acid or docohexaenoic acid, present in fish oils but not in vegetable or meat oils, are responsible for the observed reduction in autoimmunity, since both fatty acids are capable of modifying tissue and cellular cyclooxygenase metabolite levels. In addition to causing alterations in autoimmunity, the authors postulate that one or both of these unique fatty acids may decrease cyclooxygenase metabolites and protect the kidney from renal disease. Kelley, V. E., et al., J. Clin. Invest. 77:252 (1986). Using two different autoimmune mouse strains--MRL-1pr and NZBxNZW FI hybrid--having predictably progressive forms of lupus nephritis, which mimics human renal disease, the authors demonstrated an incremental increase in intrarenal TXB.sub.2 synthesis as renal function deteriorated and renal pathologic events progressed, but no consistent increases in PGE.sub.2 or 6-keto PGF.sub.1 alpha, to other cyclooxygenase metabolites, were observed. Renal disease was prevented by either pharmacologic doses of PGE.sub.2 or dietary supplementation with fish oil, in which case TXB.sub.2 did not increase.
Prior to the present invention, then, a need has existed to reduce the substantial nephrotoxic effects of cyclosporine in order to allow the use of that drug in the clinical management of transplant and nontransplant patients. It has been noted that steady state trough levels of cyclosporine must be closely maintained to be sufficiently immunosuppressive to avoid transplant rejection and still avoid nephrotoxicity and other side effects of cyclosporine, and that this narrow therapeutic window is difficult to maintain in practice. A method that would allow clinicians to widen the margin of safety between the dose of cyclosporine required to achieve effective immunosuppression and the dose likely to cause renal damage would be of great therapeutic value in the treatment of transplant patients and patients suffering from immune diseases.